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Introduction The degree and duration of response to epidermal growth factor receptor (EGFR) inhibitors in EGFR mutated lung cancer are heterogeneous. We hypothesized that the concurrent genomic landscape of these tumors, which is currently unknown in view of the prevailing single gene assay diagnostic paradigm in clinical practice, could play a role in clinical outcomes and/or mechanisms of resistance. Methods We retrospectively probed our institutional lung cancer database for tumors with EGFR kinase domain mutations that were also evaluated by more comprehensive molecular profiling, and evaluated tumor response to EGFR tyrosine kinase inhibitors (TKIs). Results Out of 171 EGFR mutated tumor-patient cases, 20 were sequenced using at least a limited comprehensive genomic profiling platform. 50% harbored concurrent TP53 mutation, 10% PIK3CA mutation, 5% PTEN mutation, among others. The response rate to EGFR TKIs, the median progression-free survival (PFS) to TKIs, the percentage of EGFR -T790M TKI resistance and survival had higher trends in EGFR mutant/ TP53 wild-type cases when compared to EGFR mutant/ TP53 mutant tumors (all p >0.05 without statistical significance); with a significantly longer median PFS in EGFR -exon 19 deletion mutant/ TP53 wild-type cancers treated with 1st generation EGFR TKIs (p=0.035). Conclusions Concurrent mutations, specifically TP53 , are common in EGFR mutated lung cancer and may alter clinical outcomes. Additional cohorts will be needed to determine if comprehensive molecular profiling adds clinically relevant information to single gene assay identification in oncogene-driven lung cancers.

In this study, we investigated the impact of initial tumor volume, rate of tumor growth, cohort size, study duration, and data analysis method on chemotherapy treatment response classifications in patient-derived xenografts (PDXs). The analyses were conducted on cisplatin treatment response data for 70 PDX models representing ten cancer types with up to 28-day study duration and cohort sizes of 3–10 tumor-bearing mice. The results demonstrated that a 21-day dosing study using a cohort size of eight was necessary to reliably detect responsive models (i.e., tumor volume ratio of treated animals to control between 0.1 and 0.42)—independent of analysis method. A cohort of three tumor-bearing animals led to a reliable classification of models that were both highly responsive and highly nonresponsive to cisplatin (i.e., tumor volume ratio of treated animals to control animals less than 0.10). In our set of PDXs, we found that tumor growth rate in the control group impacted treatment response classification more than initial tumor volume. We repeated the study design factors using docetaxel treated PDXs with consistent results. Our results highlight the importance of defining endpoints for PDX dosing studies when deciding the size of cohorts to use in dosing studies and illustrate that response classifications for a study do not differ significantly across the commonly used analysis methods that are based on tumor volume changes in treatment versus control groups.

Background The continued development of targeted therapeutics for cancer treatment has required the concomitant development of more expansive methods for the molecular profiling of the patient's tumor. We describe the validation of the JAX Cancer Treatment Profile™ (JAX-CTP™), a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable mutations in solid tumors to inform the selection of targeted therapeutics for cancer treatment. Methods NGS libraries are generated from DNA extracted from formalin fixed paraffin embedded tumors. Using hybrid capture, the genes of interest are enriched and sequenced on the Illumina HiSeq 2500 or MiSeq sequencers followed by variant detection and functional and clinical annotation for the generation of a clinical report. Results The JAX-CTP™ detects actionable variants, in the form of single nucleotide variations and small insertions and deletions (≤ 50 bp) in 190 genes in specimens with a neoplastic cell content of ≥ 10%. The JAX-CTP™ is also validated for the detection of clinically actionable gene amplifications. Conclusions There is a lack of consensus in the molecular diagnostics field on the best method for the validation of NGS-based assays in oncology, thus the importance of communicating methods, as contained in this report. The growing number of targeted therapeutics and the complexity of the tumor genome necessitate continued development and refinement of advanced assays for tumor profiling to enable precision cancer treatment.

The fragments of liturgical music held in the monastic library of Ampleforth Abbey, Yorkshire, consist of thirty-three manuscript parchment leaves of varying sizes, most of which appear to have been used as strengtheners for book bindings.1 It is difficult to determine their provenance but some clues may be found in the variety of their neume styles. Six of the fragments are written with staffless neumes, the rest have staff notation, including eleven leaves from an Office antiphoner. There are also several fragments with square notation in a mensural form. The neume styles indicate a wide time-span, probably from the eleventh century to the mid-fifteenth century. The geographical region suggested by the notation is, for the most part, that of Lorraine and of southern Germany.

Introduction: A comprehensive somatic tumor profile with associated treatment selection options requires the detection of gene fusions. After evaluating the clinical utility of multiple methods of gene fusion detection, it was determined that the Archer FusionPlex Solid Tumor Panel (AFPSTP) best compliments the JAX Cancer Treatment ProfileTM (JAX-CTPTM) clinical test in terms of workflow, specimen requirements and turnaround time. Here we describe our analytical validation process for the AFPSTP assay. Methods: AFPSTP was validated using 24 samples: 5 JAX Patient Derived Xenograft (PDX) cases, 4 translocation positive controls, 2 FFPE cancer samples, 1 normal tissue sample, and 12 cell lines. Nine of the cell lines were previously identified as positive for fusion transcripts and 3 lacked detectable fusion events. The validation was executed in 5 phases: (1) confirm that AFPSTP was able to detect known fusion or lack of fusion events in characterized specimens; (2) determine inter-assay concordance; (3) determine intra-assay concordance; (4) LOD and (5) sensitivity. Results: The fusion detection results for this validation are listed in Table 1. All but one of these fusion events was previously identified. The one novel fusion was confirmed using TaqMan RT-PCR. In addition to the expected fusions, 4 false positive events were detected, 2 due to mispriming and 2 determined to be WT read through transcripts. The fusion detection inter and intra-assay concordance was found to be 100% and the sensitivity was calculated to be 91.67% at a LOD of 5%. Conclusion: This analysis outlines the clinical validation of the incorporation of AFPSTP into the JAX-CTPTM test system. Once incorporated, the AFPSTP assay will accomplish the goal of making JAX-CTPTM a more comprehensive somatic tumor profiling assay without affecting the current acceptable turnaround time or required input material. List of 15 samples that were found to be fusion positive and the corresponding detected fusion.HorizonDx EML4/ALK PositiveEML4 → ALK variant 1HorizonDx RET PositiveCCDC6 → RETHorizonDx ROS PositiveSLC34A2 → ROS1HorizonDx Triple PositiveEML4 → ALK variant 3bHorizonDx Triple PositiveSLC34A2 → ROS1HorizonDx Triple PositiveCCDC6 → RETA673 Cell LineEWSR1 → FLI1VCaP Cell LineTMPRSS2 → ERGKM-12 Cell LineTPM3 → NTRK1RPMI-2650 Cell LineBRD4 → NUTM1NCI-H716 Cell LineFGFR2 → COL14A1OCI-AML2 Cell LineMBNL1 → RAF1REH Cell LineETV6 → RUNX1MDA-MB-175-VII Cell LineTENM4 → NRG1ASPS-1 Cell LineTFE3 → ASPSCR1ASPS-1 Cell LineASPSCR1 → TFE3PDX1EML4 → ALK 3bPDX2SYN2 → PPARG Citation Format: Samantha Helm, Aleksandra Ras, Vanessa Spotlow, Kevin Kelly, Susan Mockus, Cara Statz, Guruprasad Ananda, Joan Malcolm, Gregory J. Tsongalis. Validation of the Archer FusionPlex solid tumor panel in the JAX cancer treatment profileTM. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3630.

e23209Background: The isolation of both genomic DNA (gDNA) and total RNA (RNA) from tissues of various types is critical for comprehensive molecular profiling, especially as submitted specimens bec...

The Jackson Laboratory Cancer Treatment Profile™ (JAX-CTP™) is a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable gene variants in solid tumors to inform the selection of targeted therapeutics for cancer treatment. We will describe the design of the 358- gene panel, analytical validation, and the curation and clinical reporting of actionable variants. Selection of the gene panel was based on known drug targets, casual implications in cancer, and a thorough pathway analysis. DNA is extracted from FFPE tumor samples and using hybrid capture, the genes of interest are enriched and sequenced on Illumina HiSeq 2500 or MiSeq sequencers. FASTQ files generated from Illumina's CASAVA software are submitted to the JAX Clinical Genome Analytics (CGA) data analysis pipeline to perform automated read quality assessment, alignment, and variant calling. Identified variants are then submitted for clinical curation using a combination of the in-house JAX Clinical Knowledgebase (CKB) and the external Genetic Variant Annotation (GVA) from CollabRx. Once clinically annotated, the variants are graded relative to their clinical utility for the specific tumor type and compiled into a clinical report to inform patient treatment. Extensive analytical validation, following CAP guidelines, was conducted to assess limit of detection, accuracy, precision, sensitivity, and specificity of the assay. The summarized optimized sensitivity of the assay is a minimum coverage of samples at 300X, a limit of detection of 10% for SNP’s/indels and ≥6 copies for CNV’s, and an average of 3-4 actionable variants per sample. The challenges of interpreting gene variants for clinical actionability and for establishing an analytically valid bioinformatic pipeline will be discussed in-depth. Citation Format: Susan M. Mockus, Guruprasad Ananda, Micaela Lundquist, Vanessa Spotlow, Al Simons, Talia Mitchell, Grace A. Stafford, Christopher S. Potter, Vivek Philip, Timothy Stearns, Anuj Srivastava, Mary Barter, Lucy Rowe, Joan Malcolm, Carol Bult, Radha Krishna Murthy Katuturi, Karen Rasmussen, Douglas Hinerfeld. Design, validation, and interpretation of an NGS assay for actionable variants in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4816. doi:10.1158/1538-7445.AM2015-4816